Project Abstract Fundamental gaps exist in our understanding in Tat endolysosome escape and its role in the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). The objective here is to determine molecular mechanisms by which Tat escapes endolysosomes, and the extent to which this escape contributes to neurotoxicity. We will test our central hypothesis that Tat escapes endolysosomes through protein pores formed by the V0 sector of v-ATPase, that this escape is facilitated by calcium released from endolysosome two-pore channels (TPCs), and that such a calcium-dependent Tat endolysosome escape process, an upstream event of LTR transactivation, precedes and contributes to Tat-induced neurotoxicity. Guided by our preliminary findings, this novel hypothesis will be tested by pursuing three specific aims. (1) Determine the involvement of v-ATPase and TPCs in Tat endolysosome escape. (2) Determine the involvement of v-ATPase and TPCs in Tat-induced neurotoxicity. (3) Determine the involvement of v-ATPase and TPCs in Tat-induced learning and cognitive impairment as well as HAND-like pathology in Tat transgenic mice. The proposed work is highly innovative because it focuses on determining mechanisms by which Tat escapes endolysosomes via protein pores formed by V0 sector of v-ATPase; a process that can be regulated by calcium released from endolysosomes. Such a novel calcium-dependent Tat endolysosome escape process might be an early and upstream event of Tat-induced neurotoxicity. Successful completion of the proposed studies will provide novel insights into HIV-1 infection and the pathogenesis of HAND and may yield new and effective therapeutic strategies against HAND.